Assays are being developed in efforts to reduce the number of animals required for research. In particular, cell based systems that can evaluate a variety of compounds and/or screen single compounds with many cell types would provide the most advantageous platforms. These types of assay systems would be applicable to drug discovery, toxicology testing and environmental screening. While many cell types can be used in these systems, there are some cell types that are more relevant. Hepatocytes are one such cell type. However, their functions in culture become compromised very quickly and they have not been particularly amenable to cryopreservation when frozen as individual cells either in suspension or as a monolayer. Hepatocytes will aggregate in culture forming spheroids and studies have shown that spheroids maintain their specific functions longer in culture than freshly isolated hepatocytes. Therefore, spheroids have wide applicability for many different assay systems. Even better would be preserved spheroids that could be used whenever they were needed. To date, only one published study has been performed evaluating liver spheroids after cryopreservation. In the current study, both freezing and vitrification cryopreservation methods for liver spheroids will be developed using primary hepatocytes from rats. Freezing involves preservation with formation of ice. A protocol derived from studies cryopreserving hepatocytes will be employed. Vitrification is an ice-free preservation method that has been used successfully in some tissues. Two vitrification solutions and two rewarming methods will be evaluated using two non-specific viability assays and two liver cell-specific function assays. Either approach could prove successful and the goal of this study is to demonstrate viability and specific function of liver spheroids by either method. Phase II studies will involve optimization for the best method of cryopreservation by either freezing or vitrification in anticipation of commercialization of mammalian liver spheroids. [unreadable] [unreadable] [unreadable]